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Information on EC 1.14.19.58 - tryptophan 5-halogenase
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1.14.19.58 tryptophan 5-halogenaseIUBMB Comments
A flavin-dependent halogenase. The enzyme from the bacterium Streptomyces rugosporus catalyses halogenation of the C-5 position of tryptophan during the biosynthesis of the antibiotic compound pyrroindomycin B. It utilizes molecular oxygen to oxidize the FADH2 cofactor, giving C4a-hydroperoxyflavin, which then reacts with chloride to produce a hypochlorite ion. The latter reacts with an active site lysine to generate a chloramine, which chlorinates the substrate. cf. EC 1.14.19.59, tryptophan 6-halogenase and EC 1.14.19.9, tryptophan 7-halogenase.
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Word Map
- 1.14.19.58
- halogenases
-
flavin-dependent
-
regioselective
-
7-halogenase
-
chlorinate
- indole
- synthesis
- albogriseolus
-
biocatalysts
-
brominate
- cl
- flavin
- pharmacology
The expected taxonomic range for this enzyme is: Actinomycetia
Synonyms
tryptophan 5-halogenase, trp-5-halogenase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
mibH
pyrH
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
L-tryptophan + FADH2 + chloride + O2 + H+ = 5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SYSTEMATIC NAME
IUBMB Comments
L-tryptophan:FADH2 oxidoreductase (5-halogenating)
A flavin-dependent halogenase. The enzyme from the bacterium Streptomyces rugosporus catalyses halogenation of the C-5 position of tryptophan during the biosynthesis of the antibiotic compound pyrroindomycin B. It utilizes molecular oxygen to oxidize the FADH2 cofactor, giving C4a-hydroperoxyflavin, which then reacts with chloride to produce a hypochlorite ion. The latter reacts with an active site lysine to generate a chloramine, which chlorinates the substrate. cf. EC 1.14.19.59, tryptophan 6-halogenase and EC 1.14.19.9, tryptophan 7-halogenase.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
3-indolepropionate + FADH2 + chloride + O2 + H+
6-chloro-3-indolepropionate + 5-chloro-3-indolepropionate + FAD + 2 H2O
57% conversion by the wild-type enzyme, 75% 5-chlorination is by mutant L460F/P461E/P462T in comparison to 90% 6-chlorination by the wild-type SttH
-
-
?
anthranilamide + FADH2 + chloride + O2 + H+
5-chloro-anthranilamide + FAD + 2 H2O
43% conversion by the wild-type enzyme
-
-
?
anthranilate + FADH2 + chloride + O2 + H+
5-chloro-anthranilate + FAD + 2 H2O
1.1% conversion by the wild-type enzyme
-
-
?
indole-3-acetic acid + FADH2 + chloride + O2 + H+
5-chloroindole-3-acetic acid + FAD + 2 H2O
-
-
-
?
kynurenine + FADH2 + chloride + O2 + H+
5-chloro-kynurenine + FAD + 2 H2O
79% conversion by the wild-type enzyme
-
-
?
lantibiotic NAI-107-[tryptophan] + FADH2 + Cl- + O2 + H+
lantibiotic NAI-107-[5-chlorotryptophan] + FAD + 2 H2O
N-phenylanthranilate + FADH2 + chloride + O2 + H+
5-chloro-N-phenylanthranilate + FAD + 2 H2O
20% conversion by the wild-type enzyme
-
-
?
L-tryptophan + FADH2 + Br- + O2 + H+
5-bromo-L-tryptophan + FAD + 2 H2O
-
-
-
-
?
L-tryptophan + FADH2 + Br- + O2 + H+
5-bromo-L-tryptophan + FAD + 2 H2O
brominating activity is about 75% of the chlorinating activity
-
-
?
L-tryptophan + FADH2 + Br- + O2 + H+
5-bromo-L-tryptophan + FAD + 2 H2O
brominating activity is about 75% of the chlorinating activity
-
-
?
L-tryptophan + FADH2 + chloride + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + chloride + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
pyrroindomycin biosynthesis
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
the enzyme is involved in the biosynthesis of the antibiotic pyrroindomycin B that contains an indole ring chlorinated in the 5 position
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
the enzyme is involved in the biosynthesis of the antibiotic pyrroindomycin B that contains an indole ring chlorinated in the 5 position
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
lantibiotic NAI-107-[tryptophan] + FADH2 + Cl- + O2 + H+
lantibiotic NAI-107-[5-chlorotryptophan] + FAD + 2 H2O
NAI-107 acts as a peptidoglycan biosynthesis inhibitor by sequestering the cell wall precursor lipid II
-
-
?
lantibiotic NAI-107-[tryptophan] + FADH2 + Cl- + O2 + H+
lantibiotic NAI-107-[5-chlorotryptophan] + FAD + 2 H2O
NAI-107 acts as a peptidoglycan biosynthesis inhibitor by sequestering the cell wall precursor lipid II
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-
?
additional information
?
-
structure-based analysis of the enzyme's substrate specificity, structure-function relationship, overview
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-
?
additional information
?
-
structure-based analysis of the enzyme's substrate specificity, structure-function relationship, overview
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-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
L-tryptophan + FADH2 + Br- + O2 + H+
5-bromo-L-tryptophan + FAD + 2 H2O
-
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
pyrroindomycin biosynthesis
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
the enzyme is involved in the biosynthesis of the antibiotic pyrroindomycin B that contains an indole ring chlorinated in the 5 position
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
5-chloro-L-tryptophan + FAD + 2 H2O
the enzyme is involved in the biosynthesis of the antibiotic pyrroindomycin B that contains an indole ring chlorinated in the 5 position
-
-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
no substrate inhibition by tryptophan up to 2 mM
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.075
anthranilamide
wild-type enzyme, pH and temperature not specified in the publication
0.241
kynurenine
wild-type enzyme, pH and temperature not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.02
anthranilamide
wild-type enzyme, pH and temperature not specified in the publication
0.0085
kynurenine
wild-type enzyme, pH and temperature not specified in the publication
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.019
anthranilamide
wild-type enzyme, pH and temperature not specified in the publication
0.035
kynurenine
wild-type enzyme, pH and temperature not specified in the publication
0.01
L-tryptophan
mutant enzyme E46D, in potassium phosphate buffer (10 mM, pH 7.2), at 30°C
0.027
L-tryptophan
mutant enzyme F49A, in potassium phosphate buffer (10 mM, pH 7.2), at 30°C
0.039
L-tryptophan
mutant enzyme E46Q, in potassium phosphate buffer (10 mM, pH 7.2), at 30°C
0.05
L-tryptophan
mutant enzyme Y454F, in potassium phosphate buffer (10 mM, pH 7.2), at 30°C
0.54
L-tryptophan
wild type enzyme, in potassium phosphate buffer (10 mM, pH 7.2), at 30°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.2
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
physiological function
evolution
structure comparisons reveal subtle amino acid differences within the MibH substrate binding site generates a solvent exposed crevice presumably involved in determining the substrate specificity of this unusual peptide halogenase
evolution
-
structure comparisons reveal subtle amino acid differences within the MibH substrate binding site generates a solvent exposed crevice presumably involved in determining the substrate specificity of this unusual peptide halogenase
-
malfunction
a gene disruption mutant in the tryptophan 5-halogenase gene no longer produces the pyrroindomycin B, but still produces pyrroindomycin A, the nonhalogenated derivative
malfunction
-
a gene disruption mutant in the tryptophan 5-halogenase gene no longer produces the pyrroindomycin B, but still produces pyrroindomycin A, the nonhalogenated derivative
-
physiological function
the enzyme is involved in the biosynthesis of the antibiotic pyrroindomycin B that contains an indole ring chlorinated in the 5 position
physiological function
expression in Arabidopsis thaliana leads to the formation of 5-chlorotryptophan, 5-chloroindole-3-acetonitrile and 5-chloro-3-indole acetic acid
physiological function
MibH is an FADH2-dependent Trp halogenase, that is only active when Trp is embedded within its cognate peptide substrate deschloro NAI-107
physiological function
-
the enzyme is involved in the biosynthesis of the antibiotic pyrroindomycin B that contains an indole ring chlorinated in the 5 position
-
physiological function
-
MibH is an FADH2-dependent Trp halogenase, that is only active when Trp is embedded within its cognate peptide substrate deschloro NAI-107
-
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant His6-tagged enzyme with bound FAD, hanging drop vapour diffusion method, mixing of 22 mg/ml protein in 20 mM HEPES, pH 8.0, and 150 mM KCl, with reservoir solution containing 12.5% MPD, 12.5% PEG 3350, 0.1 M MES/imidazole, pH 6.5, and 0.09 M NPS (1:1:1 NaNO3, Na2HPO4, (NH4)2SO4), X-ray diffraction structure determination and analysis at 1.85 A resolution
sitting-drop vapor-diffusion method at 20°C, crystal structure of a tryptophan 5-halogenase (PyrH) bound to tryptophan and FAD
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
L460F/P461E/P462T
site-directed, structure-guided mutagenesis, the mutant exhibit similar activity to the wild-type SttH, with tryptophan as a substrate, but shows a complete switch in regioselectivity compared to the wild-type enzyme without impacting on catalytic efficiency: 75% 5-chlorination is observed for the substrate 3-indolepropionate with the mutant in comparison to 90% 6-chlorination of 3-indolepropionate for the wild-type SttH. SttH is more like PyrH than PrnA, with insertions present in PyrH and SttH between residues SttH 155 and 167 and a deletion between SttH 457 and 464 compared with PrnA, the mutation, the only differences evident in the active-site region between the structures of PyrH and SttH are those of PyrH residues F451, E452 and T453 and SttH L460, P461 and P462. These residues are of particular interest because they are in close proximity to the active site in PyrH and SttH, and are positioned directly above the alpha-amino acid moiety of the substrate, tryptophan. Moreover, there isa loop insertion in PrnA in this region that is suggested to contribute to its regioselectivity. Each of these residues is mutated in SttH to the corresponding residue in PyrH, that is, L460F, P461E and P462T. Individually, each mutation reduces the relative activity of the enzyme with tryptophan, but does not have a significant effect on the observed regioselectivity, with 6-chlorotryptophan remaining the major product
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His6-tagged enzyme from Escherichia coli by nickel affinity chromatography and gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
gene mibH, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of His6-tagged enzyme in Escherichia coli, coexpression of mibH with the chaperones groES/EL and gene mibS
His-tagged apoPyrH was expressed in Pseudomonas fluorescens BL915
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pharmacology
-
5-Br- and 5-Cl-tryptophan could presumably be applied as a pharmacologically attractive precursor of serotonin
synthesis
synthesis
-
use of enzyme in selective halogenation of organic compounds
synthesis
regeneration of FADH2 using flavin dehydrogenase plus formiate dehydrogenase from Candida boidiniiat 0.28 U/ml leads to about 60% increase in yield
synthesis
-
regeneration of FADH2 using flavin dehydrogenase plus formiate dehydrogenase from Candida boidiniiat 0.28 U/ml leads to about 60% increase in yield
-
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Zehner, S.; Kotzsch, A.; Bister, B.; Suessmuth, R.D.; Mendez, C.; Salas, J.A.; van Pee, K.H.
A regioselective tryptophan 5-halogenase is involved in pyrroindomycin biosynthesis in Streptomyces rugosporus LL-42D005
Chem. Biol.
12
445-452
2005
Streptomyces rugosporus (A4D0H5), Streptomyces rugosporus LL-42D005 (A4D0H5), Streptomyces rugosporus LL-42D005
brenda
Muffler, K.; Retzlaff, M.; van Pee, K.H.; Ulber, R.
Optimisation of halogenase enzyme activity by application of a genetic algorithm
J. Biotechnol.
127
425-433
2007
Streptomyces rugosporus
brenda
van Pee, K.H.; Patallo, E.P.
Flavin-dependent halogenases involved in secondary metabolism in bacteria
Appl. Microbiol. Biotechnol.
70
631-641
2006
Streptomyces rugosporus (A4D0H5)
brenda
Zhu, X.; De Laurentis, W.; Leang, K.; Herrmann, J.; Ihlefeld, K.; van Pee, K.H.; Naismith, J.H.
Structural insights into regioselectivity in the enzymatic chlorination of tryptophan
J. Mol. Biol.
391
74-85
2009
Streptomyces rugosporus (A4D0H5), Streptomyces rugosporus LL-42D005 (A4D0H5)
brenda
Muffler, K.; Kuetchou Ngnigha, A.; Ulber, R.
Bestimmung kinetischer Parameter der FADH2-abhngigen Tryptophan-5-halogenase aus Streptomyces rugosporus
Chem. -Ing. -Tech.
82
121-127
2010
Streptomyces rugosporus
-
brenda
Ortega, M.A.; Cogan, D.P.; Mukherjee, S.; Garg, N.; Li, B.; Thibodeaux, G.N.; Maffioli, S.I.; Donadio, S.; Sosio, M.; Escano, J.; Smith, L.; Nair, S.K.; van der Donk, W.A.
Two flavoenzymes catalyze the post-translational generation of 5-chlorotryptophan and 2-aminovinyl-cysteine during NAI-107 biosynthesis
ACS Chem. Biol.
12
548-557
2017
Microbispora sp. (E2IHC5), Microbispora sp. ATCC PTA-5024 (E2IHC5)
brenda
Muffler, K.; Kuetchou Ngnigha, A.; Ulber, R.
Bestimmung kinetischer Parameter der FADH2-abhngigen Tryptophan-5-Halogenase aus Streptomyces rugosporus
Chem.-Ing.-Tech.
82
121-127
2010
Streptomyces rugosporus (A4D0H5), Streptomyces rugosporus LL-42D005 (A4D0H5)
-
brenda
Shepherd, S.A.; Menon, B.R.; Fisk, H.; Struck, A.W.; Levy, C.; Leys, D.; Micklefield, J.
A structure-guided switch in the regioselectivity of a tryptophan halogenase
ChemBioChem
17
821-824
2016
Streptomyces toxytricini (E9P162)
brenda
Patallo, E.; Walter, A.; Milbredt, D.; Thomas, M.; Neumann, M.; Caputi, L.; OConnor, S.; Ludwig-Mueller, J.; van Pee, K.
Strategies to produce chlorinated indole-3-acetic acid and indole-3-acetic acid intermediates
ChemistrySelect
2
11148-11153
2017
Streptomyces rugosporus (A4D0H5)
-
brenda
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